-
ORIGINAL RESEARCHpublished: 16 December 2015doi:
10.3389/fpls.2015.01115
Edited by:David John Burritt,
University of Otago, New Zealand
Reviewed by:Ghulam Kadir Ahmad Parveez,
Malaysian Palm Oil Board, MalaysiaDavid W. M. Leung,
University of Canterbury, New Zealand
*Correspondence:Jiyu Zhang
[email protected]
Specialty section:This article was submitted to
Plant Biotechnology,a section of the journal
Frontiers in Plant Science
Received: 18 July 2015Accepted: 25 November 2015Published: 16
December 2015
Citation:Duan Z, Zhang D, Zhang J, Di H,
Wu F, Hu X, Meng X, Luo K, Zhang Jand Wang Y (2015)
Co-transforming
bar and CsALDH Genes EnhancedResistance to Herbicide and
Drought
and Salt Stress in Transgenic Alfalfa(Medicago sativa L.).
Front. Plant Sci. 6:1115.doi: 10.3389/fpls.2015.01115
Co-transforming bar and CsALDHGenes Enhanced Resistance
toHerbicide and Drought and SaltStress in Transgenic
Alfalfa(Medicago sativa L.)Zhen Duan, Daiyu Zhang, Jianquan Zhang,
Hongyan Di, Fan Wu, Xiaowen Hu,Xuanchen Meng, Kai Luo, Jiyu Zhang*
and Yanrong Wang
State Key Laborotary of Grassland Agro-Ecosystems, College of
Pastoral Agriculture Science and Technology, LanzhouUniversity,
Lanzhou, China
Drought and high salinity are two major abiotic factors that
restrict the productivityof alfalfa. By application of the
Agrobacterium-mediated transformation method, anoxidative
responsive gene, CsALDH12A1, from the desert grass Cleistogenes
songoricatogether with the bar gene associated with herbicide
resistance, were co-transformedinto alfalfa (Medicago sativa L.).
From the all 90 transformants, 16 were positive asscreened by
spraying 1 mL L−1 10% Basta solution and molecularly diagnosis
usingPCR. Real-time PCR analysis indicated that drought and salt
stress induced highCsALDH expression in the leaves of the
transgenic plants. The CsALDH expressionlevels under drought (15 d)
and salt stress (200 mM NaCl) were 6.11 and 6.87 timeshigher than
in the control plants, respectively. In comparison to the WT
plants, noabnormal phenotypes were observed among the transgenic
plants, which showedsignificant enhancement of tolerance to 15 d of
drought and 10 d of salinity treatment.Evaluation of the
physiological and biochemical indices during drought and salt
stressof the transgenic plants revealed relatively lower Na+
content and higher K+ content inthe leaves relative to the WT
plants, a reduction of toxic on effects and maintenanceof osmotic
adjustment. In addition, the transgenic plants could maintain a
higherrelative water content level, higher shoot biomass, fewer
changes in the photosystem,decreased membrane injury, and a lower
level of osmotic stress. These results indicatethat the
co-expression of the introduced bar and CsALDH genes enhanced
theherbicide, drought and salt tolerance of alfalfa and therefore
can potentially be usedas a novel genetic resource for the future
breeding programs to develop new cultivars.
Keywords: alfalfa, bar, CsALDH gene, drought stress, salt
stress, transformation
INTRODUCTION
Drought and high salinity are two major environmental factors
that limit the agriculturalproductivity. These two environmental
constraints cause excessive accumulation of aldehydesin plant cells
by inducing rapid generation of reactive oxygen species (ROS) and
graduallyaccentuate injury to leaf cell membranes during plant
growth through lipid peroxidation
Frontiers in Plant Science | www.frontiersin.org 1 December 2015
| Volume 6 | Article 1115
http://www.frontiersin.org/Plant_Science/http://www.frontiersin.org/Plant_Science/editorialboardhttp://www.frontiersin.org/Plant_Science/editorialboardhttp://dx.doi.org/10.3389/fpls.2015.01115http://creativecommons.org/licenses/by/4.0/http://dx.doi.org/10.3389/fpls.2015.01115http://crossmark.crossref.org/dialog/?doi=10.3389/fpls.2015.01115&domain=pdf&date_stamp=2015-12-16http://journal.frontiersin.org/article/10.3389/fpls.2015.01115/abstracthttp://loop.frontiersin.org/people/257231/overviewhttp://loop.frontiersin.org/people/300399/overviewhttp://loop.frontiersin.org/people/274414/overviewhttp://www.frontiersin.org/Plant_Science/http://www.frontiersin.org/http://www.frontiersin.org/Plant_Science/archive
-
Duan et al. Transform bar and CsALDH Genes in Alfalfa
(Hou and Bartels, 2014). Plants have evolved complex systemsto
respond to adverse environmental changes at physiologicaland
biochemical levels, by accumulating compatible solutesand
protective proteins, regulating ion absorption and waterbalance,
scavenging reactive oxygen, and more (Tang et al.,2014). However,
plants are negatively affected by eliciting rapidand excessive
accumulation of ROS, through regulation of theexpression of a wide
range of stress-responsive genes adapting tovarious abiotic
stress.
Aldehyde dehydrogenases (ALDHs) are NAD (P)+-dependentenzymes
are considered to be ‘aldehyde scavengeres’ to eliminatetoxic
aldehydes, due to catalyzing the irreversible oxidation of awide
range of endogenous and exogenous aromatic and aliphaticaldehydes
into corresponding carboxylic acids (Zhu et al., 2014).ALDHs are an
evolutionary conserved gene superfamily. A seriesof studies showed
that many ALDHs are capable of coping withvarious abiotic stresses
by indirectly reducing lipid peroxidationor detoxifying cellular
ROS. Overexpression of ALDH22A1 intransgenic tobacco plants
increased stress tolerance (Huanget al., 2008). Transgenic
Arabidopsis plants overexpressingn Ath-ALDH3 showed improved
tolerance to osmotic stress, heavymetals, MV, and H2O2 (Sunkar et
al., 2003). Under normalconditions, ALDH genes also play vital
roles in plant fundamentalmetabolic pathways. ALDH genes contribute
to the synthesisand catabolism of a wide of range of biomolecules
(Zhu et al.,2014).
Alfalfa (Medicago sativa L.) is an important forage legumethat
is widely grown throughout the world. Alfalfa provides highquality
of forage for animals and also improves soil fertility.However,
environmental abiotic stress, such as soil salinity andlimited
water supplies in agriculture are major constraints to
theproductivity of alfalfa. Moreover, alfalfa yield can be
reducedthrough competition with weeds for growth conditions. Its
foragequality can be lowered by decreasing the digestibility and
proteincontent of hay brought about by moderate to severe
weedinfestation (Cords, 1973; Kapusta and Strieker, 1975).
Alfalfayield from the first herbage cut can be reduced by 60–80%
whenvolunteer monocots are not controlled in the fall. The
totalalfalfa yield for a season (consisting of three to five
herbagecuts) was reduced by 25–35% (Pike and Stritzke, 1984; Ott et
al.,1989). Transgenic alfalfa plants grown in a soil medium
underglasshouse conditions were phenotypically normal and
exhibitedbialaphos resistance (Montague et al., 2007). Genetic
engineeringnow has the potential to further improve plant growth
andcrop productivity by selectively delivering genes that encodefor
proteins with known enzymatic or structural functions orregulatory
proteins involved in stress resistance traits (Newell-McGloughlin,
2014). In our previous study, a ALDH12A1 genewas cloned from
Cleistogenes songorica, a xerophytic desertgrass, and stress
inducible expression of rd29A:: CsALDH intransgenic Arabidopsis
plants showed improved tolerance todrought stress (Zhang et al.,
2014). This paper reports a studywhere the bar and CsALDH genes
were co-transformed for thefirst time intoMedicago sativa using the
Agrobacterium-mediatedmethod. The aim of our research was to
generate superior alfalfatransformation events with resistance to
herbicide and droughtor salt stress.
MATERIALS AND METHODS
Plant materials sterilized alfalfa (Medicago sativa L.
cvJinhuanghou) seeds were germinated on half-strength MSmedium in
the dark at 24◦C for 2 days and at 4◦C for 16 h priorto
transformation. The CsALDH12A1 gene from Cleistogenessongorica and
the bar gene driven by CaMV 35S promoter, wereintroduced into the
binary vector pEarlygate101.
TransformationThe Agrobacterium strain GV3101, harboring a
binaryvector pEarlygate101 bar-35S::CsALDH, was used forthe
transformation. The T-DNA region of pEarlygate101bar-35S::CsALDH is
illustrated in Supplementary Figure S1.
Overnight cultures of Agrobacterium (OD600 = 0.5) in LBmedium,
were collected by centrifugation at 5000 rpm for 15 minat 4◦C and
re-suspended in 30 ml VIM containing MS salt,Gamborg B5 vitamins
and 3% sucrose (pH 5.7) to an OD600 of0.5–0.7. The bacterial
suspension was maintained in an incubatorshaker at 80 rpm, 28◦C,
for approximately 30 min prior to use.
The transformation and transplanting process was similarto that
described by Weeks et al. (2008). The explants wereimmersed in the
Agrobacterium suspension. In addition, 0.03%(v/v) silwet77 and 1.8
g sterile white quartz sand were added toexcised seedlings and VIM
in the 50 mL centrifuge tube. Thetube was vortexed for 30 min at
the highest speed (setting at ‘8’,3200 rpm) by vertically placing
in a large sample set platformhead at room temperature to allow for
swirling of the sand inthe suspension. Treated seedlings were
vertically placed into theco-cultivation medium (0.5 MS medium,
1.5% sucrose, 0.48%agarose, pH 5.8), and supplemented with 2% (v/v)
DMSO. Petriplates were sealed with parafilm and incubated in the
chamber.Following the co-cultivation period, all seedlings were
transferredand vertically placed in SDM, consisting of 1/2 MS
medium,1.5% sucrose, 500 mg/L cefotaxime and 0.48% agarose (pH5.8).
Plates were stored in a growth chamber. The calluses
weresubcultured every 2 days. After 14 d on SDM, stably
transformedand established plantlets were transferred into plastic
culturepots (8 cm × 10 cm) containing 80 cm3 of vermiculite
andperlite (1:1), and they were acclimated to lower humidity in
anenvironmental growth chamber.
PCR and RT-PCR AuthenticationWhen the plants were at a height of
approximately 20 cm,1 mL L−1 10% Basta solution (8.0 mg l−1) was
used forpreliminary screening. The plants were sprayed three
timesevery 6 days. Genomic DNA was extracted from the alfalfaleaves
using a Plant Genomic DNA extraction kit (Tiangen,Beijing). The PCR
was conducted with a genomic DNAtemplate in PCR premix using two
convergent primers that werecomplementary to the CsALDH gene and
another pair of primersthat were complementary to the bar gene. DNA
amplification wasperformed at 94◦C for 3 min; 35 cycles of 94◦C for
30 s, 50◦Cfor 45 s, and 72◦C for 1 min 30 s, and then a final
extensionat 72◦C for 10 min. Total RNA was extracted using the
UNIQ-10 column total RNA extraction kit (Sangon Biotech,
Shanghai).The RT-PCR conditions were identical for both CsALDH
and
Frontiers in Plant Science | www.frontiersin.org 2 December 2015
| Volume 6 | Article 1115
http://www.frontiersin.org/Plant_Science/http://www.frontiersin.org/http://www.frontiersin.org/Plant_Science/archive
-
Duan et al. Transform bar and CsALDH Genes in Alfalfa
MsActin, as described in the genomic PCR analysis. The
PCRproducts were separated on a 1.2% agarose gel, stained
withGelStrain (TransGene Biotech, Beijing), and visualized underUV.
Sixteen transgenic plants showing highly expressed CsALDHwere
selected for further stress tolerance and phenotype analysis.All
subsequent experiments were conducted on cloned plantsfrom the T0
generation of transgenic plants.
Stress Treatment on Transgenic PlantsFor salt stress treatments,
plants from the transgenic and WTalfalfa lines were watered every 2
days with 1/8 Hoagland’snutrient solution for 4 weeks; then, the
nutrient solutionwas supplemented with NaCl. NaCl concentrations
wereincrementally increased by 50 mM every 2 days until the
finalconcentrations (0, 100, and 200mM)were achieved. After 10
daysof salt treatment, the plants were used for further
physiologicalanalysis.
For drought stress treatments, plants from the transgenic andWT
alfalfa lines were watered every 2 days with 1/8 Hoaglandnutrient
solution to field capacity for 4 weeks; after that, thewater was
withdrawn for15 days (all plants had severe droughtstress
symptoms). Following this, the soil was rewatered to fieldwater
capacity for 7 days. During the drought stress treatments,the
physiological indexes were measured at 0, 10, 15 days aftermoisture
stress application and 4 days after rewatering.
Expression AnalysisPlants were treated with salt stress and
drought stress to induceCsALDH expression. RNA was used to generate
first-strandcDNA with a PrimeScriptTM RT reagent Kit and gDNA
Eraser(Takara, Japan). Quantitative real-time PCR (q-RT-PCR)
wasperformed on each cDNA template using 2 × SYBR Green
PCRMasterMix (Applied Biosystems, USA) on an ABI 7500 real-timePCR
system. The transcript levels were calculated relative to
thecontrols and determined using the 2−��CT method (Zhang et
al.,2009). Data represent the means and standard errors of
threebiological replicates and two technical replicates. The
expressionlevels of the CsALDH and MsActin were analyzed by
q-RT-PCRusing the gene-specific primers listed in Supplementary
Table S1.
Phenotyping and Physiological Analysisof Transgenic PlantsPlant
height was determined by measuring the stem length fromthe top of
the shoot apex to the base of the stem under naturalgrowth
conditions. The shoot biomasses of all plants were rapidlyclipped
and weighed fresh.
The relative water content (RWC) levels were measured usingthe
procedures described by Ahmad et al. (2008). Soil watercontent
(SWC): the soil samples were dried at 105◦C until theyhad a
constant mass; then, the percentage of soil loss and dry soilmass
was used to determine the SWC.
The chlorophyll content was measured with a portablechlorophyll
meter (SPAD-502, Konica Minolta, Japan) on theintact fully expanded
fifth leaf (from the top) of individualplants. Before measurement
of the maximum quantumyield of photosystem II photochemistry
(Fv/Fm), all plants
were maintained in the dark for 30 min followed by theFv/Fm
measurement using a portable modulated chlorophyllfluorometer
(PAM-2100, Germany). The measurement wasconducted on intact fully
expanded leaves of individual plants.The net photosynthetic rate
(Pn) was measured using anautomatic photosynthetic measuring
apparatus (LI-6400, USA),as described by Qiu et al. (2003).
The malondialdehyde (MDA) content was determinedaccording to a
modified thiobarbituric acid (TBA) method(Kim and Nam, 2013). The
free proline content wasspectrophotometrically measured according
to the methodof Bates et al. (1973).
The Na+ and K+ levels were measured according to themethod
described by Flowers and Hajibagheri (2001) with
slightmodifications. The leaves from the transgenic and WT plantsof
each treatment were dried at 80◦C for 48 h, followed bydry weight
measurement. Na+ and K+ were extracted fromdried plant tissue with
100 mM acetic acid at 90◦C for at least2 h. The cation contents was
then determined using a flamespectrophotometer (2655–00, ColeParmer
Instrument Co., USA)(Bao et al., 2009).
Statistical AnalysesAll assays were biologically replicated at
least three times.The data were evaluated with Statistical Package
for the SocialSciences (SPSS 16) and Excel. The means were
separated usingDuncan’s multiple range test at p = 0.05.
RESULTS
Regeneration of Transgenic PlantspEarlygate101 bar-35S::CsALDH
was transformed intoMedicagosativa with the Agrobacterium-mediated
method. In total, 90T0 transgenic alfalfa plants after antibiotics
selection weretransplanted into pots for further analyses.
Transgenic plants were preliminarily screened by spraying1 mL
L−1 10% Basta solution. After 18 days of observing thegrowing
conditions, although most of the regenerated plantsthrived, some
transplanted, regenerated plants still had yellowcoloration or had
died; these plants may have not containedthe herbicide resistance
bar gene. Under the same conditions,the WT alfalfa died. After
spraying Basta solution, 54 transgenicplants with the CsALDH gene
were obtained and evaluated byfurther molecular authentication
(Supplementary Figure S2A).
PCR, RT-PCR, and q-RT-PCR AnalysisPutative T0 transgenic alfalfa
plants was initially screened bygenomic PCR and RT-PCR analyses. Of
the 54 plants thatsurvived, 16 displayed the diagnostic bands for
the CsALDHgene (about 1400 bp) (Supplementary Figure S2B) and bar
gene(about 450 bp) (Supplementary Figure S2C) by PCR. The
PCRpositive rate was 29.6%. The results of RT-PCR analysis
indicatedthat CsALDH was effectively expressed in all PCR
positiveplants (Supplementary Figure S2D). The expression
patternsof CsALDH during drought and salt stress were
evaluatedusing q-RT-PCR analysis. The drought and salt stress
induced
Frontiers in Plant Science | www.frontiersin.org 3 December 2015
| Volume 6 | Article 1115
http://www.frontiersin.org/Plant_Science/http://www.frontiersin.org/http://www.frontiersin.org/Plant_Science/archive
-
Duan et al. Transform bar and CsALDH Genes in Alfalfa
dramatically the increased gene expression in leaves (P <
0.05).The CsALDH expression levels in leaves with 15 d of drought
and10 d of 200 mM NaCl treatment are 6.11 times and 6.87
times,respectively, higher than the control levels (Figure 1).
Overexpression of CsALDH IncreasedDrought Tolerance in
Transgenic AlfalfaThe phenotype observation indicated that after 15
days ofdrought stress, the leaves of WT alfalfa turned yellow,
while thetransgenic alfalfa only wilted. After rewatering, the
transgenicplants recovered to the normal phenotype, while the
wild-typeplants could not be restored (Figure 2).
Plant height and shoot biomass were also measured in
thedifferent drought stress treatments. Plant height and
shootbiomass of the WT plants decreased gradually with
increaseddays in drought. While these two traits in the transgenic
plantshad little difference. In the first 10 days, the transgenic
plantsgrew normally, plant height and shoot biomass increased
slightly.There was a slight decline under the 15 days of drought
stressconditions. The transgenic plants resumed normal growth
after4 days of rewatering. These observations suggested that
theoverexpression of CsALDH confers the transformed alfalfa withan
increased drought tolerance.
To study the osmoregulatory capacity in alfalfa, the RWClevels
were evaluated during water deprivation. The RWC levelswere reduced
in both transgenic and WT plants, but the RWClevel in WT plants had
a faster decline than in transgenic alfalfaaccording to time-lapse
analysis. This observation suggested thatoverexpression of CsALDH
enhanced the capacity for osmoticadjustment of transgenic alfalfa,
resulting in greater water uptakeat low SWC during drought
stress.
To further evaluate the increased drought tolerance oftransgenic
alfalfa overexpressing CsALDH, the chlorophyllcontent, chlorophyll
fluorescence (Fv/Fm) and netphotosynthetic rate of the WT and
transgenic plants weredetermined (Figures 3A–C). Chlorophyll
fluorescence wasmonitored as a reflection of photosystem II
activity. Beforedrought stress, there was no significant (P >
0.05) difference inthe ratio of Fv to Fm (Fv/Fm) in the leaves of
WT and transgenicplants. However, after the drought treatment, the
transgenic
plants maintained high levels of Fv/Fm compared to WT plants.In
addition, although the net photosynthetic rate and
chlorophyllcontent decreased in all experimental plants with an
increase inthe drought time, the Pn and chlorophyll levels of the
WT plantshad a greater decrease compared to transgenic alfalfa.
Afterrewatering, the Pn and chlorophyll content of the
transgenicalfalfa returned to the pre-drought stress levels, while
the WTplants still exhibited a decline, indicating that the
photosystemof transgenic plants was less affected than that of the
WT plantsduring the water-deficit treatment. The photosystem of the
WTplants may have been destroyed under the extreme
droughtconditions.
The MDA content represents the degree of cell membranedamage.
Results showed that the MDA content of WT andtransgenic plants
increased after the water-deficit stress, but theMDA content of WT
plants was higher than that of transgenicplants, indicating that
the degree of membrane injury in WTplants would be higher than that
of transgenic plants (Figure 3E).When suffering from abiotic
stress, plants can accumulate morecompatible osmolytes, such as
free proline, that function asosmoprotectants so that plants can
tolerate stress. Therefore,the proline content was tested in
transgenic and WT plants(Figure 3F). Under the control conditions,
the content of freeproline was not significantly (P > 0.05)
different between theWT and transgenic plants. However, after
drought treatment,the transgenic plants accumulated higher levels
of free prolinethan the WT plants. In addition to the accumulation
of freeproline and other soluble organic osmoticum to
osmoregulation,alfalfa also adapted to drought stress by absorbing
K+ to osmoticadjustment. In this study, the contents K+ were
determinedin the leaves of transgenic plants and WT plants at
differentwater-deficit treatments (0, 10, and 15 days, and then 4
daysof rewatering) (Figure 3D). The transgenic plants
accumulatedmore K+ in their leaves, but no significant (P >
0.05) increasecould be observed in the WT plants.
Overexpression of CsALDH IncreasedSalt Tolerance in Transgenic
AlfalfaTo assess whether CsALDH is also associated with salt
stresstolerance, the transgenic and WT plants were irrigated with
0,
FIGURE 1 | Expression pattern of CsALDH gene in transgenic
alfalfa under drought and salt stress. (A) Drought stress at 0, 10,
15 days after withdrawnwatering and 4 days after rewatering; (B)
salt stress with NaCl concentrations at 0, 100, and 200 mM. P <
0.05.
Frontiers in Plant Science | www.frontiersin.org 4 December 2015
| Volume 6 | Article 1115
http://www.frontiersin.org/Plant_Science/http://www.frontiersin.org/http://www.frontiersin.org/Plant_Science/archive
-
Duan et al. Transform bar and CsALDH Genes in Alfalfa
FIGURE 2 | Drought stress tolerance of CsALDH transgenic plants.
(A) Drought stress at 0, 10, 15 days after withdrawn watering and 4
days after rewatering;(B) leaf relative water content (RWC) and
soil water content (SWC) of WT and transgenic plants; (C) plant
height of WT and transgenic plants; and (D) shoot biomassof WT and
transgenic plants. WT, wild-type plants; CsALDH, transgenic alfalfa
co-expressing bar and CsALDH. P < 0.05.
100, and 200 mM NaCl solution for 10 days. Phenotypically,
thetransgenic plants did not differ from theWT plants under
normalcontrol growth conditions. After watering with salt
solutions(100 and 200 mM of NaCl) for 10 days, the WT plants
showedchlorosis and their growth ceased under the treatments
over100 mM NaCl. However, transgenic alfalfa plants were
onlyaffected slightly; they stayed green and continued to grow.
At200 mM NaCl, the WT plants almost died while the transgenicplants
exhibited chlorosis (Figure 4A).
To further study the effect of salinity on plant height andshoot
biomass expression in the transgenic andWT alfalfa plants,these
traits were measured from the different treatments ofNaCl
concentrations. The plant height and shoot biomass of allplants
decreased gradually with increased salt concentrations.However,
expression of these two traits in the WT plants weresignificantly
(P < 0.05) lower than transgenic plants at the sameNaCl
concentration (Figures 4B,C).
The chlorophyll content, chlorophyll fluorescence (Fv/Fm)and net
photosynthetic rates of the WT and transgenic plantswere also
determined to evaluate the increased salt toleranceof transgenic
alfalfa overexpressing CsALDH (Figures 5A,B).Before salt stress,
there was no significant difference (P > 0.05)in the Fv/Fm or
chlorophyll content in the leaves of the WTand transgenic plants;
only the net photosynthetic rate showedan exceptional phenomenon in
which the Pn in transgenic plants
was lower than that inWT, which may indicate that it is
dormant.However, after the NaCl treatment, transgenic plants
maintainedhigh levels of Fv/Fm, chlorophyll content and Pn compared
tothe WT plants. Additionally, although the Fv/Fm,
chlorophyllcontent and Pn decreased in all experimental plants with
theincrease in NaCl, the Fv/Fm, chlorophyll level and Pn of
WTplants decreased more than the levels detected in the
transgenicalfalfa.
The MDA content was also examined (Figure 5C). Undercontrol
conditions, the MDA content of transgenic plants wascomparable to
that of WT plants. After 10 days of treatment withdifferent NaCl
concentration treatment (0, 100, and 200mM), theMDA content of WT
and transgenic plants increased. The MDAcontent of transgenic
plants was lower than that of WT plants.This indicated the lower
membrane injury of transgenic plants.To determine whether CsALDH
overexpression impacted on theproline content, the proline content
in the transgenic and WTplants was measured (Figure 5D). No
significant differences inthe proline content were detected between
theWT and transgenicplants at 0 mM NaCl. Significantly higher
proline was detectedwhen the transgenic plants were treated with
100 and 200 mMNaCl thanWT plants, indicating that proline was
involved in salttolerance (P < 0.05).
To ascertain whether CsALDH gene expression altered theNa+ and
K+ absorption under salt stress conditions, the
Frontiers in Plant Science | www.frontiersin.org 5 December 2015
| Volume 6 | Article 1115
http://www.frontiersin.org/Plant_Science/http://www.frontiersin.org/http://www.frontiersin.org/Plant_Science/archive
-
Duan et al. Transform bar and CsALDH Genes in Alfalfa
FIGURE 3 | Measurement of physiological changes under
drought-stress treatment. Drought stress at 0, 10, 15 days after
withdrawn watering and 4 daysafter rewatering. (A) Chlorophyll
fluorescence (Fv/Fm); (B) net photosynthetic rate (Pn); (C)
chlorophyll content; (D) K+ content; (E) MDA content; and (F)
prolinecontent. P < 0.05.
accumulation of Na+ and K+ in the NaCl-treated transgenicand WT
alfalfa plants was measured (Figures 5E,F). The NaCltreatment
significantly increased the cellular Na content in boththe
transgenic and the WT plants (P < 0.05). However, at 100and 200
mM NaCl, there was significantly more Na+ in thetransgenic plants
in comparison to the WT plants. Although theK+ levels decreased
with increasing NaCl concentrations in theleaves of both the
transgenic and WT plants, the K+ content inthe transgenic plants
was significantly (P < 0.05) higher than thatof the WT
plants.
DISCUSSION
The ALDHs superfamily consists of diverse enzymes involvedin
endogenous and exogenous aldehyde metabolism. ActiveALDHs have been
demonstrated to be required in thedetoxification of aldehydes by
oxidation to their correspondingcarboxylic acids and a large number
of ALDHs have been
shown to be involved in improvement of stress
tolerance(Rodrigues et al., 2006; Huang et al., 2008; He et
al.,2014; Zhang et al., 2014). In this study, CsALDH
wasoverexpressed in alfalfa together with the bar gene,
whichprovides herbicide resistance. The results from this
studyindicated that overexpression of CsALDH significantly (P <
0.05)enhanced the drought and salt tolerance of transgenic
alfalfaplants.
Previous studies have reported that the maintenance of
highcytosolic K+/Na+ ratios could be crucial for salt-tolerant
plants(Ren et al., 2005). The transgenic alfalfa plants,
co-expressionof ZxNHX and ZxVP1-1, resulted with more Na+, K+,
andCa2+ accumulation in leaves and roots (Bao et al., 2015).
Byanalyzing the toxic effects of ions, we found that
transgenicalfalfa overexpressing CsALDH had decreased Na+ and
increasedK+ levels, resulting in a higher K+/Na+ ratio compared to
theWT plants (Figures 5E,F). These results indicated that
CsALDHpositively regulates the salt tolerance of alfalfa by
regulating theK+/Na+ homeostasis to reduce ion toxicity.
Frontiers in Plant Science | www.frontiersin.org 6 December 2015
| Volume 6 | Article 1115
http://www.frontiersin.org/Plant_Science/http://www.frontiersin.org/http://www.frontiersin.org/Plant_Science/archive
-
Duan et al. Transform bar and CsALDH Genes in Alfalfa
FIGURE 4 | Salt stress tolerance of CsALDH transgenic plants.
salt stress with NaCl concentrations at 0, 100, and 200 mM. (A)
Growth of WT and CsALDHtransgenic plants under salt stress; (B)
plant height of WT and transgenic plants; and (C) shoot biomass of
WT and transgenic plants. P < 0.05.
FIGURE 5 | Measurement of physiological changes under
salt-stress treatment. salt stress with NaCl concentrations at 0,
100, and 200 mM. (A) Chlorophyllfluorescence (Fv/Fm); (B) net
photosynthetic rate (Pn); (C) MDA content; (D) proline content; (E)
Na+ content; and (F) K+ content. (P < 0.05).
Abiotic stress results in the excessive accumulation of ROSin
plants, leading to lipid peroxidation. Because MDA isan end-product
of lipid peroxidation in biomembranes, theMDA content reflects the
extent of lipid peroxidation andmembrane injury. Oxidative
stress-induced membrane damagehas been used as efficient criteria
for assessing the degree of
salt and drought tolerance of plants (Sairam et al., 2005;
Zhanget al., 2014). Overexpressing ScALDH21 tobacco plants
showedhigher growth ratio, higher proline accumulation, lower
MDAcontents, and stronger photosynthetic capacities, when
subjectedto drought and salt stress (Yang et al., 2015). We found
that theMDA content of the transgenic alfalfa was lower under both
salt
Frontiers in Plant Science | www.frontiersin.org 7 December 2015
| Volume 6 | Article 1115
http://www.frontiersin.org/Plant_Science/http://www.frontiersin.org/http://www.frontiersin.org/Plant_Science/archive
-
Duan et al. Transform bar and CsALDH Genes in Alfalfa
and drought stress compared to the WT plants. These resultsimply
that the degree of membrane injury of transgenic plantswas less
than that of WT plants, which is in agreement with theenhanced salt
tolerance phenotype of transgenic alfalfa.
Osmotic adjustments, by accumulating osmoprotectantsinside the
cell, are essential for reducing the cellular osmoticpotential
against an osmotic gradient between root cellsand the outside
saline solution, which eventually restoresthe water uptake into
roots during salinity stress (Li et al.,2014). Proline and soluble
sugar often osmoprotectants,allow the plants to tolerate stress. In
this study, weexamined the contents of the osmoprotectant proline
inplants. The free proline levels were higher during droughtand
salt stress conditions, and the free proline levels
inCsALDH-overexpressing transgenic plants were higher thanthose in
WT plants (Figures 3F and 5D). Therefore,the increased accumulation
of proline contributes to theincreased drought and salt tolerance
of transgenic alfalfa.Intriguingly, analysis of the K+ level of the
plants thatwere subjected to different levels of drought stress
revealedthat the transgenic plants accumulate more K+ in
theirleaves, but no significant increase could be observed in theWT
plants. This could indicate that the plants absorb K+as part of
osmotic adjustment when adapting to droughtstress.
The lack of water and salinity limit photosynthetic capacity.In
fact, the Fv/Fm ratio, a parameter commonly known asthe maximum
quantum yield of primary photochemistry orthe photochemical
efficiency of PS II (Daud et al., 2015),is used as a basic tool in
plant photosynthetic research forstress studies. The total
chlorophyll content and Pn decreasedunder saline conditions. Gama
et al. (2007) reported that areduction in plant growth is
associated with a reduction inphotosynthesis. Percival et al.
(2003) mentioned that the leafchlorophyll fluorescence responses to
increasing salinity werereduced. The reduction in Fv/Fm due to
salinity stress could berelated to chlorophyll damage under saline
conditions (Ganievaet al., 1998). Our results showed that the Fv/Fm
ratio and Pndecreased as the degree of drought and salt stress
increased, andtheir levels in CsALDH-overexpressing transgenic
plants werehigher than those in WT plants during drought and salt
stress.As a result, the increased Fv/Fm, chlorophyll content and
Pnlikely contributed to the increased stress tolerance of
transgenicalfalfa.
CONCLUSION
The exogenous CsALDH gene and bar gene were co-transformedinto
the most important forage legume crop, alfalfa, with
theAgrobacterium-mediated transformation method. The
transgenicplants grew better than the WT during drought and salt
stressconditions and, at worst, wilted slightly, whereas the growth
ofWT plants was stunted. Many WT plants did not survive
afterrewatering, while the transgenic plants resumed normal
growth.The overexpression of the CsALDH gene in the alfalfa
genomeenhanced its drought and salt tolerance through improving
plantgrowth, RWC, membrane protection, compatible
osmolytes,chlorophyll fluorescence, chlorophyll contents, and Pn.
TheCsALDH and bar transgenic alfalfa plants generated as
resistantsources in this study had improved stress and herbicide
tolerance,which could help in weed control resulting in
productioncosts and enhancing the growth of the alfalfa cultivars
duringdrought and in salty soils. However, field experiment need to
bearranged for further evaluation of stress and herbicide
tolerancein CsALDH and bar transgenic alfalfa plants.
AUTHOR CONTRIBUTIONS
Conceived and designed the experiments: JZ, YW, XH, ZD.Performed
the experiments: ZD, JZ, DZ. Ana-lyzed the data: ZD.Contributed
reagents/materials/analysis tools: ZD, KL, DZ, HD,FW, XM. Wrote the
paper: ZD.
ACKNOWLEDGMENTS
This work was financially supported by grants from NationalBasic
Research Program (973) of China (2014CB138704), theNational Natural
Science Foundation of China (31572453) andSpecial Fund for
Agro-scientific Research in the Public Interest(201403048-3). We
thank Dr. Zulfi Jahufer from AgResearch,New Zealand for kind
revising the manuscript.
SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found
onlineat:
http://journal.frontiersin.org/article/10.3389/fpls.2015.01115
REFERENCES
Ahmad, R., Kim, M. D., Back, K. H., Kim, H. S., Lee, H. S.,
Kwon, S. Y.,et al. (2008). Stress-induced expression of choline
oxidase in potato plantchloroplasts confers enhanced tolerance to
oxidative, salt, and drought stresses.Plant Cell Rep. 27, 687–698.
doi: 10.1007/s00299-007-0479-4
Bao, A. K., Du, B. Q., Touil, L., Kang, P., Wang, Q. L., and
Wang, S. M. (2015).Co-expression of tonoplast Cation/H antiporter
and H -pyrophosphatase fromxerophyte Zygophyllum xanthoxylum
improves alfalfa plant growth undersalinity, drought and field
conditions. Plant Biotechnol. J. 2012, 1–12.
doi:10.1111/pbi.12451
Bao, A. K., Wang, S. M., Wu, G. Q., Xi, J. J., Zhang, J. L., and
Wang, C. M. (2009).Overexpression of the Arabidopsis H+-PPase
enhanced resistance to salt and
drought stress in transgenic alfalfa (Medicago sativa L.). Plant
Sci. 176, 232–240.doi: 10.1016/j.plantsci.2008.10.009
Bates, L., Waldren, R., and Teare, I. (1973). Rapid
determination of freeproline for water-stress studies. Plant Soil
39, 205–207. doi: 10.1007/BF00018060
Cords, H. P. (1973). Weeds and alfalfa hay quality.Weed Sci. 21,
400–401.Daud, M. K., Quiling, H., Lei, M., Ali, B., and Zhu, S. J.
(2015).
Ultrastructural, metabolic and proteomic changes in leaves of
uplandcotton in response to cadmium stress. Chemosphere. 120,
309–320. doi:10.1016/j.chemosphere.2014.07.060
Flowers, T., and Hajibagheri, M. (2001). Salinity tolerance
inHordeum vulgare: ionconcentrations in root cells of cultivars
differing in salt tolerance. Plant Soil 231,1–9. doi:
10.1023/A:1010372213938
Frontiers in Plant Science | www.frontiersin.org 8 December 2015
| Volume 6 | Article 1115
http://journal.frontiersin.org/article/10.3389/fpls.2015.01115http://www.frontiersin.org/Plant_Science/http://www.frontiersin.org/http://www.frontiersin.org/Plant_Science/archive
-
Duan et al. Transform bar and CsALDH Genes in Alfalfa
Gama, P., Inanaga, S., Tanaka, K., and Nakazawa, R. (2007).
Physiological responseof common bean (Phaseolus vulgaris L.)
seedlings to salinity stress. Afr. J.Biotechnol. 6, 79–88.
Ganieva, R. A., Allahverdiyev, S. R., Guseinova, N. B., Kavakli,
H. I., and Nafisi, S.(1998). Effect of salt stress and synthetic
hormone polystimuline K on thephotosynthetic activity of cotton
(Gossypium hirsutum). Turk J. Bot. 22, 217–221.
He,D.H., Lei, Z. P., Xing, H. Y., and Tang, B. S. (2014).
Genome-wide identificationand analysis of the aldehyde
dehydrogenase (ALDH) gene superfamiliy ofGossypium raimondii. Gene
549, 123–133. doi: 10.1016/j.gene.2014.07.054
Hou, Q., and Bartels, D. (2014). Comparative study of the
aldehyde dehydrogenase(ALDH) gene superfamily in the glycophyte
Arabidopsis thaliana and Eutremahalophytes. Ann. Bot. 115, 465–479.
doi: 10.1093/aob/mcu152
Huang, W. Z., Ma, X. R., Wang, Q. L., Gao, Y. F., Xue, Y., Niu,
X. L., et al. (2008).Significant improvement of stress tolerance in
tobacco plants by overexpressinga stress-responsive aldehyde
dehydrogenase gene from maize (Zea mays). PlantMol. Biol. 68,
451–463. doi: 10.1007/s11103-008-9382-9
Kapusta, G., and Strieker, C. F. (1975). Selective control of
downy brome in alfalfa.Weed Sci. 23, 202–206.
Kim, G. B., and Nam, Y. W. (2013). A novel
delta(1)-pyrroline-5-carboxylatesynthetase gene of Medicago
truncatula plays a predominant role in stress-induced proline
accumulation during symbiotic nitrogen fixation. J. PlantPhysiol.
170, 291–302. doi: 10.1016/j.jplph.2012.10.004
Li, M., Guo, S., Xu, Y., Meng, Q., Li, G., and Yang, X. (2014).
Glycine betaine-mediated potentiation of HSP gene expression
involves calcium signalingpathways in tobacco exposed to NaCl
stress. Physiol. Plant. 150, 63–75. doi:10.1111/ppl.12067
Montague, A., Ziauddin, A., Lee, R., Ainley, W. M., and
Strommer, J. (2007). High-efficiency phosphinothricin-based
selection for alfalfa transformation. PlantCell Tissue Organ Cult.
91, 29–36. doi: 10.1007/s11240-007-9274-8
Newell-McGloughlin, M. (2014). “Genetically improved crops,” in
The OxfordHandbook of Food, Politics, and Society, ed. R. J.
Herring (New York: OxfordUniversity Press), 65.
Ott, P. M., Dawson, J. H., and Appleby, A. P. (1989). Volunteer
wheat (Triticumaestivum) in newly seeded alfalfa (Medicago
sativa).Weed Technol. 3, 375–380.
Percival, G. C., Fraser, G. A., and Oxenham, G. (2003). Foliar
salt tolerance of acergenotypes using chlorophyll fluorescence. J.
Arboricult. 29, 61–65.
Pike, D. R., and Stritzke, J. F. (1984). Alfalfa (Medicago
sativa)–cheat (Bromussecalinus) competition.Weed Sci. 32,
751–756.
Qiu, N., Lu, Q., and Lu, C. (2003). Photosynthesis, photosystem
II efficiency andthe xanthophyll cycle in the salt-adapted
halophyte atriplex centralasiatica. NewPhytol. 159, 479–486. doi:
10.1046/j.1469-8137.2003.00825.x
Ren, Z. H., Gao, J. P., Li, L. G., Cai, X. L., Huang, W., Chao,
D. Y., et al. (2005).Arice quantitative trait locus for salt
tolerance encodes a sodium transporter.Nat. Genet. 37, 1141–1146.
doi: 10.1038/ng1643
Rodrigues, S. M., Andrade, M. O., Gomes, A. P. S., DaMatta, F.
M., Baracat-Pereira, M. C., and Fontes, E. P. B. (2006).
Arabidopisis and tobaccoplants ecopically expression the soybean
antiquitin-like ALDH7 gene displayenhanced to drought salinity and
oxidative stress. J. Exp. Bot. 57, 1909–1918.doi:
10.1093/jxb/erj132
Sairam, R., Srivastava, G., Agarwal, S., and Meena, R. (2005).
Differences inantioxidant activity in response to salinity stress
in tolerant and susceptiblewheat genotypes. Biol. Plant. 49, 85–91.
doi: 10.1007/s10535-005-5091-2
Sunkar, R., Bartels, D., and Kirch, H. H. (2003). Overexpression
of a stress-inducible aldehyde dehydrogenase gene fromArabidopsis
thaliana in transgenicplants improves stress tolerance. Plant J.
35, 452–464. doi: 10.1046/j.1365-313X.2003.01819.x
Tang, L., Cai, H., Zhai, H., Luo, X., Wang, Z., Cui, L., et al.
(2014). Overexpressionof Glycine soja WRKY20 enhances both drought
and salt tolerance intransgenic alfalfa (Medicago sativa L.). Plant
Cell Tiss. Org. 118, 77–86. doi:10.1007/s11240-014-0463-y
Weeks, J. T., Ye, J., and Rommens, C. M. (2008). Development of
an in plantamethod for transformation of alfalfa (Medicago sativa).
Transgenic Res. 17,587–597. doi: 10.1007/s11248-007-9132-9
Yang, H., Zhang, D., Li, H., Dong, L., and Lan, H. (2015).
Ectopic overexpressionof the aldehyde dehydrogenase ALDH21 from
Syntrichia caninervis in tobaccoconfers salt and drought stress
tolerance. Plant Physiol. Biochem. 95, 83–91.
doi:10.1016/j.plaphy.2015.07.001
Zhang, J. Y., Duan, Z., Jahufer, Z., An, S. J., andWang, Y. R.
(2014). Stress-inducibleexpression of a Cleistogenes songorica ALDH
gene enhanced drought tolerancein transgenic Arabidopsis thaliana.
Plant Omics J. 7, 438–444.
Zhang, J. Y., Wang, Y. R., and Nan, Z. B. (2009). Relative and
absolutequantification expression analysis of CsSAMDC gene as a
case. ChinaBiotechnol. 29, 86–91.
Zhu, C., Chen, M., Xu, Z. S., Li, L. C., Chen, X. P., and Ma, Y.
Z. (2014).Characteristics and expression patterns of the Aldehyde
Dehydrogenase(ALDH) Gene superfamily of Foxtail Millet (Setaria
italica L.). PLoS ONE9:e101136. doi:
10.1371/journal.pone.0101136
Conflict of Interest Statement: The authors declare that the
research wasconducted in the absence of any commercial or financial
relationships that couldbe construed as a potential conflict of
interest.
Copyright © 2015 Duan, Zhang, Zhang, Di, Wu, Hu, Meng, Luo,
Zhang and Wang.This is an open-access article distributed under the
terms of the Creative CommonsAttribution License (CC BY). The use,
distribution or reproduction in other forumsis permitted, provided
the original author(s) or licensor are credited and that
theoriginal publication in this journal is cited, in accordance
with accepted academicpractice. No use, distribution or
reproduction is permitted which does not complywith these
terms.
Frontiers in Plant Science | www.frontiersin.org 9 December 2015
| Volume 6 | Article 1115
http://creativecommons.org/licenses/by/4.0/http://creativecommons.org/licenses/by/4.0/http://creativecommons.org/licenses/by/4.0/http://creativecommons.org/licenses/by/4.0/http://creativecommons.org/licenses/by/4.0/http://www.frontiersin.org/Plant_Science/http://www.frontiersin.org/http://www.frontiersin.org/Plant_Science/archive
Co-transforming bar and CsALDH Genes Enhanced Resistance to
Herbicide and Drought and Salt Stress in Transgenic Alfalfa
(Medicago sativa L.)IntroductionMaterials And
MethodsTransformationPCR and RT-PCR AuthenticationStress Treatment
on Transgenic PlantsExpression AnalysisPhenotyping and
Physiological Analysis of Transgenic PlantsStatistical Analyses
ResultsRegeneration of Transgenic PlantsPCR, RT-PCR, and
q-RT-PCR AnalysisOverexpression of CsALDH Increased Drought
Tolerance in Transgenic AlfalfaOverexpression of CsALDH Increased
Salt Tolerance in Transgenic Alfalfa
DiscussionConclusionAuthor
ContributionsAcknowledgmentsSupplementary MaterialReferences